We have recently identified the Mst1/2 ser/thr kinases as important hepatocellular carcinoma (HCC) tumor suppressors. Mst1/2 are the orthologs of the Drosophila tumor suppressor Hippo, which promotes apoptosis and inhibits cell proliferation during development. Hippo action is mediated by a set of critical substrates which are conserved in humans, and which in turn suppress the transcriptional coactivator, Yorkie, whose human ortholog, Yap, is an established oncogene and regulator of organ size. By generating Mst1/2 KO mice we found that Mst1 and Mst2 are required to maintain quiescence of liver cells and that their liver-specific ablation results in Yap activation, liver overgrowth, resistance to apoptosis, and the rapid development of HCC. Significantly, we have determined that ~30% of human HCCs have deregulation of the Mst1/2-Yap signaling axis. These results establish Mst1/2 as important tumor suppressors relevant to the pathogenesis of HCC in humans and point to Yap as one critical downstream target. Although Mst1/Mst2 lie at the center of an important growth control network, much about the physiologic regulation and functions of Mst1/2 and their role as tumor suppressors remains to be determined. We find that the upstream regulation and outputs of Mst1 and Mst2 in mammalian cells differ markedly from those predicted by the Drosophila Hippo pathway and by in vitro overexpression studies. In murine naive T cells, e.g., Mst1/2 negatively regulate proliferation independently of Yap. Moreover Mst1/2 are constitutive partners of the Rassf family of tumor suppressor polypeptides, some of which (e.g., Nore1/Rassf5) are regulated by Ras-like GTPases. Which of these multiple upstream elements control Mst1/Mst2 in liver is an open question. This proposal describes a systematic approach to define in molecular terms the upstream regulatory inputs and critical outputs of Mst1/2 that determine the capacity of Mst1/2 to regulate liver growth and exert tumor suppression in vivo. The regulation of Yap and its contribution to carcinogenesis will be investigated in detail. We have also identified Yap- independent responses to Mst1/2 deficiency, whose mechanism of activation and contribution to the malignant transformation of hepatocytes will be determined. Finally we will characterize the human HCCs that exhibit deregulation of the Mst1/Mst2 pathway, and determine how such deregulation influences the histopathologic and clinical features of human HCC. These studies are likely to reveal new strategies for the therapy of HCC and as well as of other human cancers that have defective regulation of the Mst1/2 kinases.